Gain-of-function mutations of KIT are found in a number of human malignancies, including gastrointestinal stromal tumors (GIST), mast cell neoplasms, melanoma, seminoma, and acute myeloid leukemia. GIST tumors are resistant to both radiation and chemotherapy. KIT mutations are the oncogenic driver in the case of most GISTs. Tyrosine kinase inhibitors (TKIs) such as imatinib have revolutionized the treatment of advanced GIST. In mast cell neoplasms (SM), the disease is caused by mast cells harboring activating KIT mutations. SM patients are candidates for chemotherapy, but these therapies are toxic and ineffective. Melanomas with activating KIT mutations are aggressive and do not respond to chemotherapy. In all three diseases, the effectiveness of KIT TKIs is limited by primary or secondary drug resistance. Our proposal seeks to improve therapy for KIT-mutant malignancies by identifying the role LMTK3 has in positively regulating the expression of oncogenic KIT protein. Plan: Our proposal has three specific aims. In SA1, we will determine the mechanisms by which LMTK3 regulates the transcription of oncogenic KIT isoforms. In SA2, we will determine the functional domains of LMTK3 required for regulated expression of oncogenic KIT. In SA3, we will identify proteins that interact with LMTK3 and determine their role in promoting KIT transcription. Methods: In SA1, we will use a KIT promoter-reporter construct to identify regions of the KIT promoter that are regulated by LMTK3 knockdown. In addition, we will identify candidate transcription factors that mediate indirect regulation of KIT transcription by LMTK3. In SA2, we will create a series of LMTK3 mutants with mutation of specific domains (e.g. LMTK3 kinase domain) and determine which domains are required for regulation of the KIT promoter. In addition, we will identify LMTK3 phosphorylation sites using mass spectrometry. In SA3 we will use mass spectrometry and protein pull down assays to identify proteins that interact with LMTK3 and potentially regulate KIT transcription. Clinical Relevance: The care of veterans with cancer represents a significant portion of the overall Veterans Affairs Health Care budget. New cancer treatment agents that target significant aspects of tumor biology will greatly increase the quality of life o veterans with cancer. Our study of the mechanisms by which LMTK3 regulates oncogenic KIT proteins has the potential to identify new treatments for KIT-mutant cancers. These new treatments are likely to be more effective than conventional treatments and be associated with significantly less treatment toxicity.